Extracellular vesicles transfer chromatin-like structures that induce non-mutational dysfunction of p53 in bone marrow stem cells

Small extracellular vesicle (sEV)-DNA has recently emerged as a promising biomarker for cancer diagnosis and prognosis 1 . Despite the growing interest in EV-DNA, many questions related to its nature, loading mechanism, localization, and post-shedding function(s) remain unre-vealed. Recently, we have published evidence suggesting an unequal distribution of sEV-DNA between different compartments of the recipient cells, including the nucleus 2 . This ﬁ nding motivated us to ask whether sEV-DNA is associated with proteins and what is the con-sequence of this association in the recipient cells. Although histones are abundant in sEVs 3 , whether they are free or associated with sEV-DNA and what is the effect of this association is unknown. Technically,

sEVs were stored at -80 until further use.
To isolate sEVs from DNase I containing supernatants, cells were cultivated uder the same conditions as described above in the presence of 1 ug/mL of cell culture grade DNase I (Roche, Germany).

Isolating sEVs with EdU labled EV-DNA
For EV-DNA-based functional and uptake studies, 5 µM of 5-ethynyl-2'-deoxyuridine (EdU; Thermofisher Scientific, Germany) solution was added to the CCM few hours after seeding the cells (i.e., when cells are attached to the dish-es). EdU is a thymidine analog incorporated into newly synthesized DNA during active DNA replication; thereby, cells treated with EdU release sEVs in which DNA is metabolically labeled with EdU.

Extracellular vesicles isolation from patient samples
Samples were first centrifuged for 10 min at 500 g and 4°C to remove red cells. Plasma samples (2 mL) were collected in new tubes, centrifuged at 3000 g and 4°C for 20 min, and the supernatants were stored at -80°C until use. Samples were concentrated to 500 µL in Amicon (R) Ultra -2ml centrifugal filters (Merck Millipore, Billerica, MA) at 4000 x g. Samples were washed twice with PBS onto the filter to reduce the protein content and prevent clogging. Samples were then loaded onto a qEV2 column (IZON Science, USA). sEVs' fractions were then concentrated using Amicon® Ultra-4 Centrifugal filter unit with Ultracel-10 membrane (Merck Millipore, Billerica, MA) and stored at -80 until further use.

EV-DNA characterization
Genomic DNA and EV-DNA extraction-gDNA and EV-DNA were extracted using the QIAmp DNA kit and QIAmp DNA micro kit, according to the manufacturer's instructions (QIAGEN, Germany).
After isolation, EV-DNA samples were eluted in 22 μL nuclease-free water. All DNA samples were stored at −20°C. dsDNA quantification was performed using the sensitive QuantiFluor® ONE dsDNA System (Promega, Germany), providing a fluorescent double-stranded DNA-binding dye (504nmEx/531nmEm).

Characterization of AML-EV-DNA after treatment of MV4-11 cells by DNase I during sEVs
biogenesis-EV-DNA was extracted from CCM and sEVs (Fig. S2a) and loaded onto a 1.5% agarose gel (Sigma Aldrich, Germany) and run for 75 min. DNA was then detected using highly sensitive SYBR Gold nucleic acid staining for 30 min at room temperature (Thermo Fisher Scientific, Germany).

Global DNA methylation
The global methylation profile of EV-DNA and gDNA was determined using the MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit (EpigenTek, Brooklyn, NY, USA) according to the manufacturer's instructions. The absorbance at 450 nm was assayed using a Tecan Infinit 200 Microplate Photometer (Tecan, Switzerland). Results were reported as a percentage (%) of 5-mC methylated DNA relative to the input DNA quantity, according to the following formula: 5-mC%=((Sample OD-Negativ control OD))/(Slope×Input DNA)×100

Immunoblotting
Total cells and sEV proteins were determined using BCA and micro BCA assay kits (Invitrogen).
Concentrated vesicle suspensions and cell lysates were then treated with 4X Laemmli buffer (Biorad, Germany) in the presence of beta-Mercaptoethanol and protease and phosphatase inhibitors cocktail at 95°C for 10 min. Samples were then loaded on NuPAGE 4-12 % Gel (Invitrogen, Germany), resolved for 2h (100 V), transferred onto the Immuno-blot PVDF membrane (Merck Millipore), and subsequently blocked with 5% dry milk (Roth, Germany) in TBS-T (Tris Buffered Saline with 0.1% Tween-20) for 1 hour. The membranes were then incubated overnight at 4°C with the primary antibodies listed in Supplementary Table 1. The blots were vigorously washed whit TBS-T and then incubated with the corresponding secondary antibodies for 90 min at room temperature. Membranes were detected with Pierce ECL plus Western blotting substrate (Thermo Fisher Scientific), and images were taken on Fusion FX Machine (Vilber Lourmat Deutschland GmbH).

Transmission electron microscopy
Negative staining was performed at the Electron Microscopy Unit (EMU) of the Imaging Center Essen (IMCES) for sEVs. In addition, FBS18 EV was included as a negative control. Briefly, 3 µl of sEVs were added onto a Formvar-coated 200 mesh copper grid (#SF162, PLANO GmbH) which had a hydrophilic surface due to being exposed to glow discharging for 1.5 minutes (easiGlow™, PELCO).
Excess liquid was removed, and the grids were allowed to dry for at least 2 minutes. Samples were observed using a JEOL JEM-1400 Plus TEM (JEOL) at 120 kV, and the images were processed using ImageJ to determine the average diameter.

Bead-assisted flow cytometry
sEVs were analyzed by flow cytometry for semi-quantitative detection of sEV protein CD81 according to the protocol we described before 1 . Briefly, sEVs were incubated with aldehyde-sulfate latex beads (4 µM; Invitrogen) for 30 min at RT. After removing the unbound beads, 5% BSA was added for blocking for 30 min at RT. Blocked sSEVs-Beads were then stained with CD81-FITC (Beckman Coulter, Marseille, France). Data were acquired in conventional flow cytometers (BD FACS Aria, BD Biosciences, Heidelberg, Germany) and analyzed using FlowJo™ v10.8 Software (BD Life Sciences).

Chromatin Immuno-precipitation followed by deep sequencing (ChIP-Seq)
ChIP assay was performed using the ab500 chromatin immunoprecipitation kit (Abcam Biotechnology, MA, USA). Cells were seeded onto T175 flasks according to the doubling time. Cells were then scraped and washed twice with DPBS, and aliquots of 1 × 10 7 were considered for the next ChIP steps. Cells and sEVs (100 µL) were cross-linked in 1% of formaldehyde (ThermoFisher, Germany) for 10 min at room temperature and subsequently neutralized with glycine. Cells and EVs were lysed Sheared chromatins were then incubated with ChIP grade rabbit polyclonal anti-dsDNA antibody (Abcam Biotechnology, MA, USA) overnight at 4°C on a rotating wheel. The antibodychromatin mixture was incubated with Protein A beads for 1 h at 4°C, and DNA was purified using the abcam DNA slurry as described before.

ChIP Mass spectrometry and data analysis
Sample preparation, LC-MS/MS, data processing, and data analysis were performed at the EMBL Proteomics Core Facility (Heidelberg, Germany) according to the following protocol: Sample preparation-Reduction of disulphide bridges in cysteine containing proteins was performed with dithiothreitol (56°C, 30 min, 10 mM in 50 mM HEPES, pH 8.5). Reduced cysteines were alkylated with 2-chloroacetamide (room temperature, in the dark, 30 min, 20 mM in 50 mM HEPES, pH 8.5). Samples were prepared using the SP3 protocol 2,3 and trypsin (sequencing grade, Promega) was added in an enzyme to protein ratio 1:50 for overnight digestion at 37°C. Next day, peptide recovery in HEPES buffer by collecting supernatant on magnet and combining with second elution wash of beads with HEPES buffer. Peptides were further cleaned up using an OASIS® HLB µElution Plate (Waters) according to manufacturer's instructions.

Data analysis-
The raw output file of MaxQuant (ProteinGroups.txt -file) was processed using the R programming language (ISBN 3-900051-07-0). As a quality filter, only proteins were allowed that were quantified with at least two unique peptides. Raw iBAQ values were used without normalization. Differential expression was evaluated by computing the respective ratio of raw iBAQ values. In order to try to annotate the ratio (coming from a single replicate) with a p-value, the ratio distribution was assumed to come from a student's t-distribution from which p-values were estimated using the 'pt' function from R. The degrees of freedom were simplified by the number of observed proteins. The false discovery rates calculated from the p-values using the 'p.adjust' function from R. This method was used to get a quick approximation.

Atomic force microscopy
To study DNA-protein association, sheared genomic chromatin and EV-chromatin were analyzed by AFM using Dimension Icon equipped with ScanAsyst FastScann head (Bruker, Germany).

Cryo-EM analysis
For cryo-EM examination, samples were vitrified using a Vitrobot Mark V (Thermo Fisher, Hilsboro Oregon) plunging device. 3 µL of the sample dispersion was applied to a Quantifoil or a lacey carbon coated TEM grid that had been glow discharged in an oxygen plasma cleaner (Diener Nano®, Diener electronic, Germany) shortly before. After removing the excess sample solution with filter paper, the grid is immediately plunged into liquid ethane. The specimen is transferred to a TEM (FEI Titan Krios G4) for the subsequent examination, keeping cryogenic conditions. Conventional TEM imaging was done using an acceleration voltage of 300 kV. Micrographs were acquired with a 4k Direct Electron Detection Camera (Gatan K3) under low-dose conditions.

Packaging EV-DNA and EV-chromatin in polymersomes
The polymersomes preparation was carried out as follows. The block copolymer polybutadiene-b-poly(ethylene ethyl phosphate) (PB-b-PEEP) was prepared as described previously 6 .
For the blank polymersomes, 20 µL of a (PB(1,4)73-b-PEEP12) solution in CHCl3 (4 mg/mL) was added to a 2 mL glass vial and concentrated in a desiccator under reduced pressure until the solvent was evaporated. An invisible thin film of the neat polymer was thus obtained. Next, 200 µL of PBS was quickly added, and the reaction was left to stir overnight (1250 min-1, 30h) vigorously. The vesicles were prepared as described above for the encapsulation experiments, adding EV-DNA or EV-chromatin to the PBS solution. The prepared vesicles were stored in a refrigerator at 4 °C until the subsequent use. Cells were fixed and permeabilized as we have described before 1 . EdU click-it reaction was carried out using Click-iT™ EdU Alexa Fluor 647 Imaging kit (ThermoFisher, Germany) following the manufacturer's instructions. Images were acquired in confocal microscopy (Leica TCS SP8) in the corresponding channels (blue-DAPI, green-H2B-GFP, and red-EdU). ImageJ was used to quantify red and green mean fluorescence intensity. EV-DNA uptake by BM-MSCs-After sEVs isolation from MV4-11 supernatants, sEVs were resuspended in FBS18 culture media and added to BM-MSCs in culture at approximately 50:1 ratio (sEVs/recipient cells). Cells fixation and permeabilization, as well as the click reaction, were performed as we have previously described.

Transcription inhibition by Actinomycin D
For transcription inhibition, BM-MSCs were first incubated with EV-DNA or EV-chromatin for 24h and then treated with 10 µg/mL of Actinomycin D (Sigmaaldrich, Germany) for 0, 1, and 2 h. Cells were then lysed for RNA extraction and the inhibition of transcription was determined with RT-qPCR.

MDM2 inhibition
Siremadlin treatment-Siremadlin HDM201 was synthesized by Global Discovery Chemistry at Novartis. For in vitro treatment, 2×10 5 BM-MSCs were first cultured in the presence of 5 µM Siremadlin.